User:Chloe Paul/Sandbox 1

Introduction
Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA, its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family), RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). The structure of RTP has been shown to be integral to it's function. RTP must be able to bind DNA (and therefore must be positively charged) and bind asymetrically (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.

The Structure of RTP
RTP has been found to exist as a symmetric α+β protein in solution and a homomeric dimer through crystal structure determination .These identical monomers each contain four α helices (α1, α2, α3, α4), one β strand (β1) and two β ribbons(β2 and β3). Int also contains a distorted N-terminal region. When the two monomers come together and the two α4 helices bind, forming a dimer with an overall rectangle shape of 66Å x 35 Å x 30 Å. The long C-terminal helices (α4) facilitate binding by establishing a hydrophobic core between the monomers (residues 93-103.) The helices form an antiparallel coiled-coil structure and additionally contribute an amino acid to the hydrophobic core of the other monomer (residue 122). Both monomers still remain structurally similar when they form the dimer in solution. It should be noted that when in solution the flexible loop between β2 and β3 are able to assume different conformation. This later gives rise to the "wing-up, wing-down" conformation when bound to native DNA .

RTP binding to DNA
RTP has been of interest in terms of its specific binding to DNA because it doesn’t use the common DNA structural motifs such as a basic leucine zipper, zinc finger or helix-turn-helix motif. It has been established that RTP, like Tus, is sequence specific, as it binds as the Ter sites. These comprise two sequences that are imperfect inverted repeats. This means that RTP needs to be able to recognise specific bases in the helical DNA structure by reading the exposed edges of the bases located in the major and minor grooves of DNA. Structurally RTP interacts with DNA through the α3 helices in the major grooves, its anti-parallel β-sheets (β2 and β3) in the minor grooves. The flexible N-terminal regions wrap with non-specific ionic interactions around the DNA. 

The Asymetric binding
The RTP:DNA interaction has been shown to be able to induce two different conformations of RTP depending upon the nature of the DNA. Early experiments used to determine how they interacted, used symmetric DNA (sDNA) which resulted in RTP binding symmetrically. However in nature, RTP was found to have a polar mechanism which implied asymetric binding, leading to further investigations of how RTP bound to DNA. It was later shown that when RTP bound to native or non-symmetric DNA (nDNA) it induced an asymmetric "wing-up, wing-down" form of RTP with a two faces. One face, known as the “blocking” face acts to terminate the approaching replication fork. The other face is described as the “permissive” face as it allows the replication fork to proceed along the DNA. These faces correspond to the A site and B site of the Ter sequence of DNA respectively. These DNA sites are the two halves of the pseudosummetric palendromic sequence. The conformation and thus function of the RTP monomer depends on which site the RTP monomer binds to. It is the concept of these two faces that give rise to the polar mechanism of RTP.

Termination Mechanism
As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction. This evidence alows us to move from a simple "fork arrest model" to a more complex understanding of termination.

Further Directions
RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity) (Ref). Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP. Investigations in to their comparative function have shown that the substitution of RTP for Tus in the E.coli system, will demonstrate no phenotypic difference, and hence share the same function as replication terminators. However the question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.